scholarly journals Precise label-free quantitative proteomes in high-throughput by microLC and data-independent SWATH acquisition

2016 ◽  
Author(s):  
Jakob Vowinckel ◽  
Aleksej Zelezniak ◽  
Artur Kibler ◽  
Roland Bruderer ◽  
Michael Muelleder ◽  
...  
Keyword(s):  
High Throughput ◽  
Flow Rate ◽  
Quantitative Proteomics ◽  
High Sensitivity ◽  
Data Consistency ◽  
Biological Research ◽  
Label Free ◽  
Key Technology ◽  
Sample Series ◽  
Acquisition Strategies

AbstractWhile quantitative proteomics is a key technology in biological research, the routine industry and diagnostics application is so far still limited by a moderate throughput, data consistency and robustness. In part, the restrictions emerge in the proteomics dependency on nanolitre/minute flow rate chromatography that enables a high sensitivity, but is difficult to handle on large sample series, and on the stochastic nature in data-dependent acquisition strategies. We here establish and benchmark a label-free, quantitative proteomics platform that uses microlitre/minute flow rate chromatography in combination with data-independent SWATH acquisition. Being able to largely compensate for the loss of sensitivity by exploiting the analytical capacities of microflow chromatography, we show that microLC-SWATH-MS is able to precisely quantify up to 4000 proteins in an hour or less, enables the consistent processing of sample series in high-throughput, and gains quantification precisions comparable to targeted proteomic assays. MicroLC-SWATH-MS can hence routinely process hundreds to thousands of samples to systematically create precise, label free quantitative proteomes.

scholarly journals High-throughput Raman-activated cell sorting in the fingerprint region

2021 ◽  
Author(s):  
Matthew Lindley ◽  
Julia Gala de Pablo ◽  
Jorgen Walker Peterson ◽  
Akihiro Isozaki ◽  
Kotaro Hiramatsu ◽  
...  
Keyword(s):  
Real Time ◽  
Information Content ◽  
High Throughput ◽  
Cell Sorting ◽  
Biological Research ◽  
Cell Populations ◽  
Label Free ◽  
Trade Off ◽  
Chemical Content ◽  
Fingerprint Region

Cell sorting is the workhorse of biological research and medicine. Cell sorters are commonly used to sort heterogeneous cell populations based on their intrinsic features. Raman-activated cell sorting (RACS) has recently received considerable interest by virtue of its ability to discriminate cells by their intracellular chemical content, in a label-free manner. However, broad deployment of RACS beyond lab-based demonstrations is hindered by a fundamental trade-off between throughput and measurement bandwidth (i.e., cellular information content). Here we overcome this trade-off and demonstrate broadband RACS in the fingerprint region (300 - 1,600 cm-1) with a record high throughput of ~50 cells per second. This represents a 100x throughput increase compared to previous demonstrations of broadband fingerprint-region RACS. To show the utility of our RACS, we demonstrate real-time label-free sorting of microalgal cells based on their accumulation of carotenoids and polysaccharide granules. These results hold promise for medical, biofuel, and bioplastic applications.

scholarly journals proDA: Probabilistic Dropout Analysis for Identifying Differentially Abundant Proteins in Label-Free Mass Spectrometry

2020 ◽  
Author(s):  
Constantin Ahlmann-Eltze ◽  
Simon Anders
Keyword(s):  
Mass Spectrometry ◽  
Quantitative Proteomics ◽  
Statistical Power ◽  
Missing Values ◽  
Ad Hoc ◽  
Linear Models ◽  
Statistical Tests ◽  
High Sensitivity ◽  
Small Sample ◽  
Label Free

Abstract Protein mass spectrometry with label-free quantification (LFQ) is widely used for quantitative proteomics studies. Nevertheless, well-principled statistical inference procedures are still lacking, and most practitioners adopt methods from transcriptomics. These, however, cannot properly treat the principal complication of label-free proteomics, namely many non-randomly missing values. We present proDA, a method to perform statistical tests for differential abundance of proteins. It models missing values in an intensity-dependent probabilistic manner. proDA is based on linear models and thus suitable for complex experimental designs, and boosts statistical power for small sample sizes by using variance moderation. We show that the currently widely used methods based on ad hoc imputation schemes can report excessive false positives, and that proDA not only overcomes this serious issue but also offers high sensitivity. Thus, proDA fills a crucial gap in the toolbox of quantitative proteomics.

scholarly journals High-Throughput Label-Free Isolation of Heterogeneous Circulating Tumor Cells and CTC Clusters from Non-Small-Cell Lung Cancer Patients

Cancers ◽  
2020 ◽  
Vol 12 (1) ◽  
pp. 127 ◽  
Author(s):  
Mina Zeinali ◽  
Maggie Lee ◽  
Arthi Nadhan ◽  
Anvya Mathur ◽  
Casey Hedman ◽  
...  
Keyword(s):  
Lung Cancer ◽  
Small Cell Lung Cancer ◽  
High Throughput ◽  
Cell Lung Cancer ◽  
High Sensitivity ◽  
Small Cell ◽  
Fish Analysis ◽  
Solid Organ ◽  
Label Free ◽  
Small Cell Lung

(1) Background: Circulating tumor cell (CTC) clusters are emerging as clinically significant harbingers of metastases in solid organ cancers. Prior to engaging these CTC clusters in animal models of metastases, it is imperative for technology to identify them with high sensitivity. These clusters often present heterogeneous surface markers and current methods for isolation of clusters may fall short. (2) Methods: We applied an inertial microfluidic Labyrinth device for high-throughput, biomarker-independent, size-based isolation of CTCs/CTC clusters from patients with metastatic non-small-cell lung cancer (NSCLC). (3) Results: Using Labyrinth, CTCs (PanCK+/DAPI+/CD45−) were isolated from patients (n = 25). Heterogeneous CTC populations, including CTCs expressing epithelial (EpCAM), mesenchymal (Vimentin) or both markers were detected. CTCs were isolated from 100% of patients (417 ± 1023 CTCs/mL). EpCAM− CTCs were significantly greater than EpCAM+ CTCs. Cell clusters of ≥2 CTCs were observed in 96% of patients—of which, 75% were EpCAM−. CTCs revealed identical genetic aberrations as the primary tumor for RET, ROS1, and ALK genes using fluorescence in situ hybridization (FISH) analysis. (4) Conclusions: The Labyrinth device recovered heterogeneous CTCs in 100% and CTC clusters in 96% of patients with metastatic NSCLC. The majority of recovered CTCs/clusters were EpCAM−, suggesting that these would have been missed using traditional antibody-based capture methods.

scholarly journals proDA: Probabilistic Dropout Analysis for Identifying Differentially Abundant Proteins in Label-Free Mass Spectrometry

2019 ◽  
Author(s):  
Constantin Ahlmann-Eltze ◽  
Simon Anders
Keyword(s):  
Mass Spectrometry ◽  
Quantitative Proteomics ◽  
Statistical Power ◽  
Missing Values ◽  
Ad Hoc ◽  
Linear Models ◽  
Statistical Tests ◽  
High Sensitivity ◽  
Small Sample ◽  
Label Free

AbstractProtein mass spectrometry with label-free quantification (LFQ) is widely used for quantitative proteomics studies. Nevertheless, well-principled statistical inference procedures are still lacking, and most practitioners adopt methods from transcriptomics. These, however, cannot properly treat the principal complication of label-free proteomics, namely many non-randomly missing values.We present proDA, a method to perform statistical tests for differential abundance of proteins. It models missing values in an intensity-dependent probabilistic manner. proDA is based on linear models and thus suitable for complex experimental designs, and boosts statistical power for small sample sizes by using variance moderation. We show that the currently widely used methods based on ad hoc imputation schemes can report excessive false positives, and that proDA not only overcomes this serious issue but also offers high sensitivity. Thus, proDA fills a crucial gap in the toolbox of quantitative proteomics.

An Automated Pipeline for High-Throughput Label-Free Quantitative Proteomics

2013 ◽  
Vol 12 (4) ◽  
pp. 1628-1644 ◽  
Author(s):  
Hendrik Weisser ◽  
Sven Nahnsen ◽  
Jonas Grossmann ◽  
Lars Nilse ◽  
Andreas Quandt ◽  
...  
Keyword(s):  
High Throughput ◽  
Quantitative Proteomics ◽  
Label Free ◽  
Automated Pipeline

scholarly journals Quantitative proteomics identification of seminal fluid proteins in male Drosophila melanogaster

2018 ◽  
Author(s):  
Irem Sepil ◽  
Ben R Hopkins ◽  
Rebecca Dean ◽  
Marie-Laëtitia Thézénas ◽  
Philip D Charles ◽  
...  
Keyword(s):  
Drosophila Melanogaster ◽  
Quantitative Proteomics ◽  
Reproductive Tract ◽  
Seminal Fluid ◽  
High Sensitivity ◽  
Female Reproductive Tract ◽  
Label Free ◽  
Seminal Fluid Proteins ◽  
Copy Numbers ◽  
Before And After

AbstractSeminal fluid contains some of the fastest evolving proteins currently known. These seminal fluid proteins (Sfps) play crucial roles in reproduction, such as supporting sperm function, and – particularly in insects – modifying female physiology and behaviour. Identification of Sfps in small animals is challenging, and often relies on samples taken from the female reproductive tract after mating. A key pitfall of this method is that it might miss Sfps that are of low abundance due to dilution in the female-derived sample or rapid processing in females. Here we present a new and complimentary method, which provides added sensitivity to Sfp identification. We applied label-free quantitative proteomics to Drosophila melanogaster male reproductive tissue – where Sfps are unprocessed, and highly abundant – and quantified Sfps before and immediately after mating, to infer those transferred during copulation. We also analysed female reproductive tracts immediately before and after copulation to confirm the presence and abundance of known and candidate Sfps, where possible. Results were cross-referenced with transcriptomic and sequence databases to improve confidence in Sfp detection. Our data was consistent with 124 previously reported Sfps. We found 8 high-confidence novel candidate Sfps, which were both depleted in mated versus unmated males and identified within the reproductive tract of mated but not virgin females. We also identified 31 more candidates that are likely Sfps based on their abundance, known expression and predicted characteristics, and revealed that four proteins previously identified as Sfps are at best minor contributors to the ejaculate. The estimated copy numbers for our candidate Sfps were lower than for previously identified Sfps, supporting the idea that our technique provides a deeper analysis of the Sfp proteome than previous studies. Our results demonstrate a novel, high-sensitivity approach to the analysis of seminal fluid proteomes, whose application will further our understanding of reproductive biology.

Development of Algorithms for Mass Spectrometry-based Label-free Quantitative Proteomics*

2011 ◽  
Vol 38 (6) ◽  
pp. 506-518 ◽  
Author(s):  
Wei ZHANG ◽  
Ji-Yang ZHANG ◽  
Hui LIU ◽  
Han-Chang SUN ◽  
Chang-Ming XU ◽  
...  
Keyword(s):  
Mass Spectrometry ◽  
Quantitative Proteomics ◽  
Label Free

Next-Generation Sequencing: An Emerging Tool for Drug Designing

2019 ◽  
Vol 25 (31) ◽  
pp. 3350-3357 ◽  
Author(s):  
Pooja Tripathi ◽  
Jyotsna Singh ◽  
Jonathan A. Lal ◽  
Vijay Tripathi
Keyword(s):  
Next Generation Sequencing ◽  
High Throughput ◽  
Large Scale ◽  
Massively Parallel Sequencing ◽  
Genomic Research ◽  
Biological Research ◽  
Next Generation ◽  
Human Welfare ◽  
Drug Designing ◽  
Generation Sequencing

Background: With the outbreak of high throughput next-generation sequencing (NGS), the biological research of drug discovery has been directed towards the oncology and infectious disease therapeutic areas, with extensive use in biopharmaceutical development and vaccine production. Method: In this review, an effort was made to address the basic background of NGS technologies, potential applications of NGS in drug designing. Our purpose is also to provide a brief introduction of various Nextgeneration sequencing techniques. Discussions: The high-throughput methods execute Large-scale Unbiased Sequencing (LUS) which comprises of Massively Parallel Sequencing (MPS) or NGS technologies. The Next geneinvolved necessarily executes Largescale Unbiased Sequencing (LUS) which comprises of MPS or NGS technologies. These are related terms that describe a DNA sequencing technology which has revolutionized genomic research. Using NGS, an entire human genome can be sequenced within a single day. Conclusion: Analysis of NGS data unravels important clues in the quest for the treatment of various lifethreatening diseases and other related scientific problems related to human welfare.

scholarly journals From Multiplex Serology to Serolomics—A Novel Approach to the Antibody Response against the SARS-CoV-2 Proteome

Viruses ◽  
2021 ◽  
Vol 13 (5) ◽  
pp. 749
Author(s):  
Julia Butt ◽  
Rajagopal Murugan ◽  
Theresa Hippchen ◽  
Sylvia Olberg ◽  
Monique van Straaten ◽  
...  
Keyword(s):  
Antibody Response ◽  
High Throughput ◽  
Large Population ◽  
High Sensitivity ◽  
Population Based ◽  
Antibody Responses ◽  
Hek293 Cells ◽  
Novel Approach ◽  
Assay Performance ◽  
Multiplex Serology

The emerging SARS-CoV-2 pandemic entails an urgent need for specific and sensitive high-throughput serological assays to assess SARS-CoV-2 epidemiology. We, therefore, aimed at developing a fluorescent-bead based SARS-CoV-2 multiplex serology assay for detection of antibody responses to the SARS-CoV-2 proteome. Proteins of the SARS-CoV-2 proteome and protein N of SARS-CoV-1 and common cold Coronaviruses (ccCoVs) were recombinantly expressed in E. coli or HEK293 cells. Assay performance was assessed in a COVID-19 case cohort (n = 48 hospitalized patients from Heidelberg) as well as n = 85 age- and sex-matched pre-pandemic controls from the ESTHER study. Assay validation included comparison with home-made immunofluorescence and commercial enzyme-linked immunosorbent (ELISA) assays. A sensitivity of 100% (95% CI: 86–100%) was achieved in COVID-19 patients 14 days post symptom onset with dual sero-positivity to SARS-CoV-2 N and the receptor-binding domain of the spike protein. The specificity obtained with this algorithm was 100% (95% CI: 96–100%). Antibody responses to ccCoVs N were abundantly high and did not correlate with those to SARS-CoV-2 N. Inclusion of additional SARS-CoV-2 proteins as well as separate assessment of immunoglobulin (Ig) classes M, A, and G allowed for explorative analyses regarding disease progression and course of antibody response. This newly developed SARS-CoV-2 multiplex serology assay achieved high sensitivity and specificity to determine SARS-CoV-2 sero-positivity. Its high throughput ability allows epidemiologic SARS-CoV-2 research in large population-based studies. Inclusion of additional pathogens into the panel as well as separate assessment of Ig isotypes will furthermore allow addressing research questions beyond SARS-CoV-2 sero-prevalence.

scholarly journals Integrating high-performing electrochemical transducers in lateral flow assay

2021 ◽  
Author(s):  
Antonia Perju ◽  
Nongnoot Wongkaew
Keyword(s):  
High Performance ◽  
Point Of Care ◽  
Low Cost ◽  
High Sensitivity ◽  
Lateral Flow ◽  
Analytical Performance ◽  
Label Free ◽  
High Performing ◽  
Lateral Flow Assays ◽  
Electrochemical Transducers

AbstractLateral flow assays (LFAs) are the best-performing and best-known point-of-care tests worldwide. Over the last decade, they have experienced an increasing interest by researchers towards improving their analytical performance while maintaining their robust assay platform. Commercially, visual and optical detection strategies dominate, but it is especially the research on integrating electrochemical (EC) approaches that may have a chance to significantly improve an LFA’s performance that is needed in order to detect analytes reliably at lower concentrations than currently possible. In fact, EC-LFAs offer advantages in terms of quantitative determination, low-cost, high sensitivity, and even simple, label-free strategies. Here, the various configurations of EC-LFAs published are summarized and critically evaluated. In short, most of them rely on applying conventional transducers, e.g., screen-printed electrode, to ensure reliability of the assay, and additional advances are afforded by the beneficial features of nanomaterials. It is predicted that these will be further implemented in EC-LFAs as high-performance transducers. Considering the low cost of point-of-care devices, it becomes even more important to also identify strategies that efficiently integrate nanomaterials into EC-LFAs in a high-throughput manner while maintaining their favorable analytical performance.

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